1. Field of the Invention
The present invention relates to the field of water quality control, bioremediation, biomonitoring and biocide treatment.
2. Description of Related Art
A small subset of the micro-organisms present in ecosystems is harmful to humans. These harmful micro-organisms are known as pathogens. Once in a stream, lake, or estuary, these harmful micro-organisms can infect humans through contaminated fish and shellfish, skin contact or ingestion of water. Aerosols are another mode of human contact with water-borne pathogens. Defective and improperly designed or installed septic tanks, sewage sludge land applications, and undetected sewage system leakage are major sources for pathogens in groundwater.
Pathogenic organisms are typically monitored by following non-pathogenic bacteria that are usually associated with the pathogenic organisms. These associated bacteria are called indicator organisms. Certain indicator organisms, referred to as fecal indicators, indicate the possible presence of human pathogenic organisms. These indicator organisms may also be used to assess the degree of pathogen removal by treatment processes or to detect contamination of distribution systems.
A commonly used indicator organism is coliform bacteria. Coliform bacteria include total coliforms and fecal coliforms. The term “total coliforms” includes several genera of gram-negative, facultative anaerobic, non-spore-forming, rod-shaped bacteria, some of which occur naturally in the intestinal tract of animals and humans, as well as others that occur naturally in soil and in fresh or marine waters and could be pathogenic to a variety of specific hosts. Fecal coliforms, including Escherichia coli (“E. coli”), are found in the intestines and feces of warm-blooded animals. Thus, the presence of E. coli in a water sample indicates fecal contamination and the possible presence of pathogens.
Efforts to reduce the amount of fecal coliforms in surface and ground waters are hindered by the uncertainty about the sources of fecal coliforms found in such waters and by an inability to link excess fecal contamination with specific sources such as sewer line leaks or wastewater treatment plant (WWTP) effluent.
Most fecal indicators are indirect and only warn of the possibility of the presence of fecal pathogens, which are not necessarily from humans, and potentially from multiple sources. This is because there are multiple bacterial species and multiple strains of a given bacterial species in the environment that can mask attempts to track a specific population of bacteria. Further, determining what happens to the micro-organisms once they reach a body of water is often as challenging as identifying and tracking their sources. As living organisms, micro-organisms require certain conditions to survive, grow, and reproduce. Many factors influence the die-off rate of viruses, bacteria, and protozoans in the environment. These factors include, but are not limited to, sunlight, temperature, moisture conditions, salinity, soil conditions, water body conditions, settling, association with particles, and encystation. Thus, there is a need for a method to unequivocally determine sources of fecal contamination and to easily track the movement of fecal contaminants in the environment. More broadly, there is a need for a method to easily track the movement and viability of micro-organisms in the environment.
One approach to identifying and tracking micro-organisms and sources of fecal contamination uses dyes or other non-living indicators. However, dyes do not necessarily behave like micro-organisms in the environment. For example, dyes do not grow and cannot reflect whether environmental conditions would have killed a living organism. In addition, dyes do not necessarily adsorb in a manner identical to micro-organisms.
A number of methodologies designed to identify the source of fecal pollution have been collectively referred to as Bacterial Source Tracking (BST) methods. BST methods provide information as to what animal the bacteria came from and not the physical source. Based on the information that BST methods provide, water quality restoration efforts can be targeted to the appropriate physical source (i.e. sewage system vs. agricultural runoff).
The BST methods can be separated into two groups based on whether the methods analyze differences in bacterial phenotype, such as outer membrane protein profiles or multiple antibiotic resistance patterns, or differences in bacterial genotype, such as restriction fragment length polymorphism, randomly amplified polymorphic DNA, or ribotyping.
All BST methods have one or more limitations to their utility in identifying specific, sources of fecal contamination. First, with all BST methods, classification of a bacterial sample as being from a human or a nonhuman source is typically reported as a probability statement, although it is unclear what minimum probability is predictive of the actual source. (Wiggins, 1996; Parveen et al., 1997; Sinton et al., 1998; Hagedom et al., 1999; Parveen et al., 1999; Wiggins et al., 1999; Bernhard and Field, 2000). Second, all BST methods are predicated on the assumption that there are differences among bacterial strains originating from human versus nonhuman sources. For example, to unambiguously identify the animal source of an unknown E. coli isolate, there must be a DNA sequence unique to that animal's E. coli isolate and the DNA sequence must be found in all E. coli isolates from that animal's species. However, genetic principles indicate that there is not a barrier to exchange of DNA between E. coli strains, regardless of animal source. As a result, definitive uniform DNA sequence differences between strains of E. coli should not exist when based solely on the strain's animal source. Second, many bacterial tracking or identification methods rely on being able to culture bacteria from a sample prior to performing the assay. Because most bacteria from environmental samples are in a not readily culturable form (e.g., the viable but nonculturable (VBNC) state), these culture-based assays will not detect these bacteria. Third, many BST methods examine populations of bacteria and do not follow specific bacteria; this can affect the interpretation of data obtained from an assay. For example, it is difficult to determine if bacteria present in an environmental sample originated from a putative contamination point source, or from a source located between the putative point source and the collection point (i.e. it cannot be determined what percentage of bacteria in a sample originated from the potential point source). BST methods are also typically very costly and time intensive to conduct. Because of the limitations of current methods in identifying and monitoring sources of fecal contamination with respect to specificity, accuracy, cost, and time, a need exists for a method to directly identify the physical source of contamination in a water system. More broadly, the need exists for a method to easily track the movement of and viability of micro-organisms in the environment.